Folic Acid-Anchored PEGgylated Phospholipid Bioconjugate and Its Application in a Liposomal Immunodiagnostic Assay for Folic Acid

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• 作者：Ja-an Annie Ho ; Chi-Hsiang Hung ; Li-Chen Wu ; Ming-Yuan Liao
• 刊名：Analytical Chemistry
• 出版年：2009
• 出版时间：July 15, 2009
• 年：2009
• 卷：81
• 期：14
• 页码：5671-5677
• 全文大小：194K
• 年卷期：v.81,no.14(July 15, 2009)
• ISSN：1520-6882

文摘

A folic acid-anchored, poly(ethylene glycol)-linked (PEGgylated) phospholipid and an immunoaffinity chromatographic column were prepared and employed to develop a liposomal immunodiagnostic assay for the direct determination of folic acid (FA) in this study. Distearoylphosphatidylethanolamine−poly(ethylene glycol)₂₀₀₀−folic acid (DSPE-PEG₂₀₀₀-FA) was synthesized through carbodiimide-mediated coupling of FA and DSPE-PEG₂₀₀₀-amine and characterized using thin layer chromatography, ¹H nuclear magnetic resonance spectroscopy, and electrospray ionization-mass spectrometry. Liposomal biolabels were constructed using the synthesized DSPE-PEG₂₀₀₀-FA in conjunction with other phospholipids. A stationary phase having affinity for FA was prepared by covalently linking purified anti-FA monoclonal antibodies onto N-hydroxysuccinimide-activated Sepharose beads, which were subsequently packed into a 1.9 cm diameter polypropylene column. The calibration curve for FA had a linear range from 10⁻⁸ to 10⁻⁴ M. The limit of detection was 6.8 ng (equivalent to 500 μL of 3.1 × 10⁻⁸ M FA). The elution buffer (35% methanol in Tris buffered saline containing 0.1% Tween 20) also served as the regeneration buffer, which allowed the same column to be used for up to 50 times without any observable loss of reactivity. The immunoaffinity chromatographic column was reusable and capable of concentrating analytes from sample solution; in conjunction with folic acid-sensitized liposomal biolabels, however, they hold great potential as sensitive immunoaffinity assays for the determination for FA. To confirm the feasibility of using this system in the analysis of real samples, the folic acid contents of three over-the-counter vitamin supplements were tested. The recoveries of folic acid of 90−112% for these three samples were obtained, suggesting contents that were consistent with the information obtained from their nutritional facts panels.